1. Field of the Invention
This invention relates to a method of using bacteriophage-encoded endolysins in the production of fuel alcohols by fermentation to control the growth of non-preferred, contaminating microorganisms such as Lactobacillus during fermentation. The LysA, LysA2, LysgaY and λSa2 endolysin constructs are active under fermentation conditions with regard to pH and presence of ethanol and lyse untreated, live lactobacilli in the fermentation cultures. LysA and λSa2 expressed in recombinant fermentation yeast Saccharomyces cerevisiae are functional and lyse lactobacilli in fermentation conditions.
2. Description of the Relevant Art
The fuel ethanol industry has experienced rapid growth in recent years, with 10.6 billion gallons produced in 2009 and future need estimated to be 60 billion gallons (340.69 billion liters) by 2030 in the United States alone. Currently, the majority of ethanol is produced from renewable carbohydrate-rich feedstock such as corn-starch or sugarcane, but to achieve higher demands in the future, lignocellulosic biomass will need to be utilized. Weakening the economics of biofuel production, are ethanol losses due to bacterial contamination of fermentation cultures. Contributing to this concern is the fact that it is not feasible to produce fuel ethanol under aseptic conditions, therefore chronic and acute contaminations are commonplace (Connolly, C. 1997. In: The Alcohol Textbook, 3rd Edition. Eds: Jacques et al., Nottingham University Press, pages 317-334; Skinner and Leathers. 2004. J. Ind. Microbiol. Biotechnol. 31: 401-408; Schell et al. 2007. Bioresource Tech. 98:2942-2948; Beckner et al. 2011. Lett. Appl. Microbiol. 53:387-394). A variety of Gram positive and Gram negative bacteria have been isolated from commercial fuel ethanol production facilities (Skinner and Leathers, supra; Beckner et al., supra; Bischoff et al. 2009. Biotech. Bioengineer. 103:117-122). However, it is generally believed that lactic acid bacteria are the most detrimental, with Lactobacillus species predominating (Beckner et al., supra; Bischoff et al., supra; Limayem et al. 2011. J. Environ. Science Health B 46(8):709-714). Lactobacilli thrive in industrial fermentation environment because they are well adapted for survival under the high ethanol, low pH and low oxygen conditions. A major culprit, L. fermentum, has been shown to reduce ethanol production in Saccharomyces cerevisiae fermentation cultures by as much as 27% (Bischoff et al., supra; Chang et al. 1995. J. Microbiol. Biotech. 5:309-314). Controlling lactic acid bacteria in fermentation cultures often requires prophylactic antibiotic treatments and/or costly production shutdowns for extensive cleaning and disinfecting (Beckner et al., supra; Lushia and Heist. 2005. Ethanol Producer Magazine May: 80-82; Narendranath, N.V. In: The Alcohol Textbook, 4th Edition. 2003. Eds.: Jacques et al., Nottingham University Press, Nottingham, Pages 287-298). Despite current control measures and practices, long-term suppression of microbial contamination is still a major challenge in ethanol production.
There are numerous theories to account for the effect contaminants have on yeast during ethanol production. Chronic lactic acid bacteria contaminants are believed to compete for sugars available for conversion to ethanol as well as essential micronutrients required for optimal yeast growth. Acute contaminations often lead to the accumulation of major inhibitory end-products such as acetic and lactic acids that inhibit yeast growth and, if left untreated, cause “stuck” fermentations (Beckner et al., supra; Makanjuola et al. 1992. Enzyme Microb. Tech. 14:350-357; Narendranath et al. 1997. Appl. Environ. Microbiol. 63:4158-4163). Besides lowering the pH of the fermentation below the optimal S. cerevisiae pH range for the conversion of sugars to ethanol, the actual inhibitory impact of the acetic and lactic acid end products has been postulated to result from the undissociated form of the acid that is capable of diffusing through the yeast cell membrane where it dissociates, acidifying the yeast cytoplasm (Schnurer and Magnusson. 2005. Trends Food Sci. Technol. 16:70-78). Other compounds produced by lactic acid bacteria are known to contribute to the inhibition of ethanol production, for example, diacetyl (Lindgren and Dobrogosz. 1990. FEMS Microbiol. Rev. 7:149-163), fatty acids (Sjogren et al. 2003. Appl. Environ. Microbiol. 69:7554-7557) and the broad spectrum antibiotic reuterin (Schnurer and Magnusson, supra; Magnusson et al. 2003. FEMS Microbiol. Lett. 219:129-135).
Several techniques are currently being employed in an attempt to control microbial contaminants. In the United States, bacterial contaminants are commonly controlled with the commercially available antibiotics virginiamycin, penicillin, and erythromycin (Beckner et al., supra; Lushia and Heist, supra; Narendranath, N.V, supra). Treatment for contamination is often prophylactic, necessitating the addition of antibiotics to each fermentation cycle. However, decreased susceptibility to virginiamycin has already been observed in Lactobacillus species isolated from dry-grind ethanol plants that use virginiamycin (Demeester and Rondelet. 1976. J. Antibiotics 29:1297-1305), and the emergence of isolates with multidrug resistance to both virginiamycin and penicillin have also been reported (Lushia and Heist, supra; Bischoff et al. 2007. J. Ind. Microbiol. Biotechnol. 34:739-744). In addition, concerns over the potential for antibiotic residues to persist in the distillers grains co-products may further limit their use during ethanol production (McChesney, D. G. 2010. FY 2010 Nationwide Survey of Distillers Grains for Antibiotic Residues, US Food and Drug Administration). A ‘no-antibiotic’ approach has obvious advantages, but acceptable alternatives are currently lacking.
Bacteriophage (phage) endolysins are lytic enzymes produced by bacterial viruses. During phage infection of bacteria, lysins are produced near the end of the phage replication cycle to degrade peptidoglycan, (PG) (a major structural component of the bacterial cell wall), leading to cell lysis (‘lysis from within’) and phage progeny release (reviewed in Bernhardt et al. 2002. Res. Microbiol. 153:493-501). Scientists have found that externally lysin-treated Gram positive bacteria still lyse (exolysis or ‘lysis from without’). Such exolysis has been exploited to control pathogenic and problematic bacteria (Loeffler et al. 2001. Science 294:2170-2172; Nelson et al. 2001. Proc. Natl. Acad. Sci. USA 98:4107-4112; Schuch et al. 2002. Nature 418:884-889; Schmelcher et al. 2012. Appl. Environ. Microbiol. 78:2297-2305). For review, see Nelson et al. 2012. Adv. Virus. Res. 83:299-365). Currently, such lysins are only considered exolytic for Gram positive bacteria because Gram negative bacteria have an outer membrane which prevents access of the lysin to the peptidoglycan of the Gram negative bacterial cell wall. However, Briers at al. and Lavigne at al. have reported using endolysin fusion proteins comprising peptides having membrane- or LPS-disrupting activity to facilitate endolysin activity (US2011/0243915 and US2012/0189606, respectively). Lysins exert their lethal effects by forming holes in the peptidoglycan. This degradation of the cells wall results in the extrusion of the cytoplasmic membrane due to the ˜30 or 40 atm intracellular pressure resulting in osmolysis (Nelson et al., supra). Peptidoglycan is unique to bacteria and has a complex structure comprising a sugar backbone of alternating units of N-acetyl glucosamine and N-acetyl muramic acid (Schleifer and Kandler. 1972. Bacteriol. Rev. 36:407-477). Typically, these sugar polymers are cross-linked by species-specific oligopeptide attachments at the N-acetyl muramic acid residues (FIG. 1a). Phages have evolved lysins to be modular in design to compensate for peptidoglycan complexity, generally consisting of both lytic domains and cell wall binding (CWB) domains (FIG. 1b). Catalytically, a single lysin molecule should be sufficient to cleave an adequate number of bonds to lyse a bacterial call (Fischetti, V. A. 2010. Int. J. Med. Microbiol. 300:357-362).
There is a need for new specific antimicrobial treatments to control lactic acid bacterial contamination in fuel ethanol fermentations. We have identified lactobacillus bacteriophage endolysins which have exolytic activity towards ˜60% of the lactobacilli tested, including four L. fermentum isolates from fuel ethanol fermentations.